Comparative Ribo-Seq of Staphylococcus aureus harboring A2058-unmodified ribosomes and m6A2058-modified ribosomes
Ontology highlight
ABSTRACT:
ORGANISM(S): Staphylococcus aureus
PROVIDER: GSE168265 | GEO | 2024/01/16
REPOSITORIES: GEO
Similar Datasets
Project description:The high-throughput sequencing has become a standard tool for analyzing gene expression. Usually a cDNA/DNA library is constructed with 5' and 3' linkers and then sequenced. Unlike mRNA, small RNA often contains modifications including 5' cap or triphosphate and 3' methylation, affecting the linker addition during cloning processes. Small RNA is expressed at much lower levels than mRNA, making it more difficult to clone small RNA using a small amount of total RNA. Here we have developed a new strategy to clone modified/unmodified small RNA in an all-liquid-based reaction in a single PCR tube using as little as 20 ng total RNA. The 7-hour cloning process only needs ~1 hour labor time. Moreover, this method can be used to clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA. Since the linkers used are derived from the Illumina Truseq linkers for DNA cloning, the PCR primers based on the linker sequences can be used to obtain amplicons derived from small RNA/mRNA/DNA. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, accurate and versatile. Moreover, the all-liquid-based reaction can be performed in an automated manner.
2020-01-24 | GSE129664 | GEO
Project description:Stalling of ribosomes due to consecutive proline motifs during polypeptide synthesis is a challenge faced by organisms across all kingdoms. To overcome this, bacteria employ translation elongation factor P (EF-P), while archaea and eukaryotes rely on a/eIF5A. Typically, these elongation factors become active only after undergoing post-translational modifications (PTMs) such as ß-lysinylation, (deoxy-)hypusinylation, rhamnosylation, or 5-aminopentanolyation. An exception to this rule is found in EF-P members of the PGKGP-subfamily, which remain unmodified. However, the mechanism behind the ability of certain bacteria to bypass metabolically and energetically costly PTMs, thus retaining active EF-P, remains unclear. In this study, we investigated the design principles governing the full functionality of unmodified EF-Ps in Escherichia coli. We first screened for naturally unmodified EF-Ps that are active in an E. coli reporter strain. We identified EF-P from Rhodomicrobium vannielii capable of rescuing the growth deficiencies and changes in the proteome of E. coli ΔepmA mutant lacking the gene for the modifying EF-P-(R)-ß-lysine ligase. We then identified specific amino acids in domain I of the unmodified EF-P variant that affected its activity. Ultimately, we transferred these functional properties to other marginally active members of the PGKGP EF-P subfamily, resulting in fully functional unmodified variants in E. coli. These results have not only implications for the improved heterologous expression of polyproline-containing proteins in E. coli but offer applications for other bacterial hosts. Understanding the mechanisms that underlie the functionality of unmodified EF-P provides insights into cellular adaptations to optimize protein synthesis.
2024-04-08 | PXD044929 | Pride
Project description: Not available
| S-EPMC6955225 | biostudies-literature
Project description:The biotin labeled DNA:RNA hybrids with or without RNA m5C modification were incubated with lysates of 293 cells treated with H2O2, and were pulled down by Streptavidin. The proteins pulled down by the empty beads control, unmodified or m5C-modified hybrids were analyzed by mass spectrometry
2022-04-29 | MSV000089337 | MassIVE
Project description:The ribosome has considerably increased in size during metazoan evolution in the form of an RNA shell that could serve as a platform for yet unknown protein interactions. Here, we have comprehensively identified the mammalian ‘ribo-interactome’ by establishing a ribosome affinity purification method. Our findings reveal a multitude of novel ribosome interacting factors, encompassing unanticipated functional categories including energy metabolism, cell redox homeostasis, as well as key protein and RNA modifying enzymes. These findings led us to characterize ufmylation, a novel posttranslational modification on ribosomes, and define its substrates. We further show that pyruvate kinase, a key enzyme for stem cell and cancer metabolism, is an RNA binding protein that unexpectedly interacts with specialized sub-pools of ribosomes at the endoplasmic reticulum (ER) and coordinates the localization and translation of ER destined mRNAs. Collectively, these studies uncover that the ribo-interactome imbues a new layer of regulatory potential in translating the genome.
2017-06-01 | GSE96998 | GEO
Project description:According to the Canadian Food Inspection Agency and Health Canada, genetically modified crops are considered safe if they are substantially equivalent to a conventional crop in regards to agronomic, physiological and compositional characteristics. A recurring issue in safety assessment of genetically modified crops is the paucity of analytical methods to detect unintended or unexpected outcomes of genetic modification. Traditional targeted compound comparative analyses are limited in scope and capacity to detect unintended changes in chemical composition. This study explored the potential of using microarray technology to assess the substantial equivalence of gene expression profiles between genetically modified and conventional soybean cultivars. Different pre processing methods were applied to the raw expression data from the arrays, and clustering methods were used to try and differentiate the genetically modified cultivars from the conventional cultivars. Results showed that more variation existed between different strains of conventional cultivars than between conventional and genetically modified cultivars. For more information, please see: Cheng, K.C., Beaulieu, J., Iquira, E., Belzile, F.J., Fortin, M.G. and Strömvik, M.V. (2008). âEffect of transgenes on global gene expression in soybean is within the natural range of variation of their conventional counterparts.â Journal of Agricultural and Food Chemistry (in press) Experiment Overall Design: Five samples (biological replicates) of total RNA from each of the five different soybean varieties were selected for hybridization to Affymetrix Soybean GeneChips, for a total of 25 chips (following total RNA integrity assessment). Spike controls B2, bio-B, bio-C, bio-D and Cre-x were added to each hybridization cocktail. Arrays were washed and stained in an Affymetrix Fluidics Station prior to scanning on the Affymetrix GeneChip Scanner 3000. Image acquisition and processing was done with the Affymetrix Microarray Analysis Suite 5.0.
2008-06-16 | E-GEOD-9374 | biostudies-arrayexpress
Project description:Histone methylation occurs on both lysine and arginine residues and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD domain (PHDUHRF1), an important regulator of DNA CpG methylation, as an unanticipated histone H3 unmodified arginine 2 (H3R2)-recognition modality. This conclusion is based on binding studies and co-crystal structures of the PHDUHRF1 bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1’s ability to repress its direct target gene expression is dependent on PHDUHRF1 binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function. UHRF1 protein was depleted in HCT116 cells by shRNA treatment. Total RNA was purified and used to determine the global gene transcription profiles by microarray assays. The UHRF1-regulated genes were identified by comparing the gene expression profiles of control and UHRF1-depleted HCT116 cells.
2011-07-07 | E-GEOD-30478 | biostudies-arrayexpress
Project description:We used RNA-seq to determine transcriptional profiles of whole flies raised on either a glucose-enriched or unmodified holidic diet.
2020-03-20 | GSE147222 | GEO
Project description:A unified strategy to clone modified/unmodified small RNAs and mRNAs for high throughput sequencing
| PRJNA532412 | ENA
Project description:In this study, we systematically identified RNAs associated with ribosomes. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a or Rpl16b, expressed under control of thier native promoter, were affinity purified from whole cell extracts of cultures grown to mid-log phase in minimal medium. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. We performed two biological replicates with each protein and analyzed the RNA content using oligo microarrays. Total RNA isolated from extracts of cells expressing Rpl16a-ZZ or Rpl16b-ZZ (input) and from the affinity-purified ribosomes was reverse transcribed with oligo(dT) primers. cDNA was labeled with Cy3 and Cy5 fluorescent dyes, respectively, and competitively hybridized to yeast oligo microarrays. Alternatively, RNA was isolated from sucrose gradient fractions containing 60S subunits, 80S monosomes and polysomes. Here, we performed four biological replicates. Analysis was done with oligo microarrays as described above. A strain or line experiment design type assays differences between multiple strains, cultivars, serovars, isolates, lines from organisms of a single species. strain_or_line_design
2010-06-25 | E-GEOD-13654 | biostudies-arrayexpress